TGF-( superfamily signaling regulates pre-adipocyte determination and differentiation in vitro. These secreted ligands play roles in the stimulation and inhibition of adipogenesis. The determination and subsequent differentiation of precursor cell lines into adipocytes is stimulated by bone morphogenetic proteins (BMPs) 2, 4 and 7. BMP7-induced adipocyte differentiation can be inhibited by GDF8/myostatin, another TGF-( superfamily member. TGF-( is also antagonistic to adipogenesis in vitro and in vivo. We hypothesize that GDF3, another TGF-( superfamily member and the subject of this proposal, also plays an important role in regulating adipogenesis and/or in affecting the function of mature adipocytes. First, GDF3 is expressed in adipose tissues and is significantly up regulated in white adipose depots in response to high fat diets, with disproportionate increases in the adipocyte and stromal vascular fractions. Secondly, over expression of GDF3, in vivo, by adenoviral transduction results in a greater sensitivity to the adipogenic effects of high fat diets, reflected by greater weight gain, adipose tissue mass, higher leptin levels, and greater adipocyte cell size. Conversely, our studies of Gdf3-/- mice have revealed a protection from diet- induced obesity, including normal body and adipose tissue mass and adipocyte cell sizes, despite a greater caloric intake. Therefore, GDF3 may function in to regulate adipogenesis and/or mature adipocyte function. The aims of the application are as follows: Specific aim 1- Examine the effects of GDF3 on cell proliferation, survival, and differentiation. We will examine the effects of exogenous GDF3 protein on the proliferation, mitotic clonal expansion, apoptosis, and differentiation of two adipogenic cell lines. Specific aim 2- Assess GDF3's effects on mature adipocyte function. Fully differentiated 3T3-L1 cells will be analyzed to determine if exogenous GDF3 protein administration has effects on lipogenesis, lipolysis, and other mature adipocyte functions. Specific aim 3- Define the essential components of GDF3 signaling in adipocytes and determine if there is a link to brown/white adipocyte fate decisions. We will further define GDF3's interactions with its receptors and with other superfamily members that affect adipogenesis. Specific aim 4- Examine the consequences of GDF3 over expression in adipose tissues in vivo. We will generate transgenic mice which express GDF3 selectively in adipose tissues under the reversible control of the drug, doxycycline.